从人类获得变性 Tenocyte 的一种协议
1Department of Orthopaedic Surgery, CHA Bundang Medical Center,CHA University School of Medicine, 2Department of Pathology, Kyung Hee University Hospital at Gangdong,Kyung Hee University School of Medicine, 3Department of Physiology,CHA University School of Medicine, 4Department of Rehabilitation, CHA Bundang Medical Center,CHA University School of Medicine
Summary
体外应用退行性肌腱细胞是必要的, 当调查的效果, 新的治疗肌腱。然而, 大多数研究研究只使用动物模型或健康的 tenocyte。我们提出了以下的协议, 以隔离人类退行性肌腱细胞在手术期间。
Abstract
肌腱, 一种因肌腱变性而发展起来的痛苦状态, 由于体力活动的增加和预期寿命的延长, 在发达世界中的上升。尽管患病率不断上升, 但潜在的发病机制仍不清楚, 治疗一般是有症状的。最近, 研究了多种治疗方法, 包括生长因子、干细胞和基因治疗, 以期提高退行性肌腱的愈合效力。然而, 这些研究中的大多数只对动物模型或健康的人类肌腱细胞进行。尽管一些研究使用病理学肌腱细胞, 我们最好的知识目前还没有描述如何获得人类退行性肌腱细胞的协议。本研究的目的是描述获取人类退行性肌腱细胞的标准协议。最初, 肌腱组织是在手术期间从侧髁的病人身上收获的。然后从腕桡肌肌腱中取出活检标本, 并与手术时观察到的结构变化有关。所有收获的肌腱看起来都是钝的、灰色的、易碎的和水肿的, 这使得它们在视觉上与健康的不同。肌腱细胞被培养并用于实验。同时, 在组织学上分析了一半的收获组织, 并表明它们具有相同的肌腱 (angiofibroblastic 发育不良或增生) 的主要特征。免疫细胞化学的二次分析证实, 培养的干细胞是肌腱细胞与大多数细胞有阳性染色的莫霍克和 tenomodulin 蛋白。用增殖试验或 qRT PCR 方法对细胞与健康对照进行比较, 确定肌腱细胞退行性的质量。退行性 tenocyte 显示出较高的增殖率和类似基因表达模式的肌腱, 匹配以前的报告。总的来说, 这一新的议定书可能为今后研究肌腱提供了有用的工具。
Introduction
肌腱是一种慢性退行性肌肉骨骼的情况, 发展在身体的各个部分。最近, 由于越来越多地参与娱乐运动和提高预期寿命1、2, 发达世界的肌腱病例数量大大增加。肌腱的病因被认为是多因素的, 这些原因包括缺血、氧自由基损伤、收缩与血管扩张 innervations 的不平衡、内微泪和神经调节的变化3 ,4,5,6,7,8。大多数治疗肌腱只会缓解症状。此外, 没有组织再生的治疗需要很长的时间来恢复和获得有限的反应肌腱, 这给医生9的临床挑战。
目前治疗方案的无能以及缺乏退行性肌腱的自愈能力, 使研究人员对探索替代治疗策略感兴趣。最近, 新的研究报告了许多有希望的结果, 以提高肌腱肌腱的愈合效果, 使用生长因子, 干细胞为主的治疗, 和基因治疗10,11,12。
通过文献综述, 我们发现, 所涉及的研究可分为两类, 根据其分析材料: 动物模型, 如鼠, 一只老鼠, 或兔子;和人类模型。关于动物模型, 目前有两种常用的技术来产生肌腱: 化学诱导损伤或机械超载的模型。然而, 每个动物模型是有限的复制复杂的人类肌腱病理13,14。
大多数使用人体标本的论文都是在组织学上分析的, 或者是以健康的人类 tenocyte 为基础进行体外实验, 而不是变性 tenocyte15、16、17、18,19,20,21. 只有少数论文报告说, 他们使用了人类退化 tenocyte, 但他们没有详细描述用于从人类22、23获得变性 tenocyte 的协议。在这方面, 应该指出, 动物模型或健康组织/tenocyte 的成功结果不一定能预测人的疗效或有效剂量, 因为肌腱变性是一个复杂的过程, 而发病机制是仍未完全理解。
总的来说, 有必要描述从人体组织获得退行性 tenocyte 的标准协议, 而不会对捐献者造成不良影响。本文介绍了如何获得人类退行性 tenocyte 的协议。为验证该协议, 对所采集的组织进行组织学分析。然后, 采用免疫细胞化学 (ICC)、定量的实时聚合酶链反应 (qRT PCR) 和活性测定方法, 证实了 tenocyte 的退化性。
Protocol
该议定书是根据《赫尔辛基宣言》进行的, 《议定书》是在茶 Bundang 医疗中心的机构审查委员会批准的。 1. 退行性肌腱组织从患者中收获 诊断侧髁的病史和使用体检结果: 压痛在腱的起源接近侧髁;疼痛通过腕关节的伸展而诱发。 使用以下纳入标准:18 岁以上;侧髁的历史至少六月;非手术治疗顽固;自注射皮质类固醇、富含血小板血浆或肉毒杆菌毒素以来, 至少有?…
Representative Results
组织学分析表明, 从侧髁的收获组织具有 tendinopathic 肌腱的特征。肌腱退行性肌腱的 H & E 段揭示了一种无条理的胶原束, 极性和细直、强包装的平行纤维结构。在样品中常见的组织学征象如高细胞构成、大核无典型纺锤形等。此外, 退化肌腱的胶原束是由 H & E 染色的相对较弱的阿利新, 但呈阳性染色, 表明软骨和多糖物质增加。 <p class="jove_content" fo:keep-together.within-page="1"…
Discussion
以前的一些研究报告了如何使用不同的程序, 如胶原酶或 kartogenin 注射, 跑步机跑步, 以及更多的26,27建立慢性 tendinopathic 动物模型。虽然许多研究显示有希望的治疗效果的基础上这些动物模型, 使用人类退行性 tenocyte 的实验将是至关重要的肌腱领域, 以重现疗效的治疗。在这篇文章中, 我们建立了一个成功地收获和文化的人类退化肌腱细胞一致的协议。?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国卫生技术研发 & 发展研究所 (KHIDI) 的资助, 该项目由大韩民国卫生部 & 福利部提供资金 (赠款号: HI16C1559)。
Materials
| Scalpel | Kisanbio | KS-Q0306-15 | No. 15 |
| Mini-blade | Beaver | 374769 | |
| Dulbecco's modified Eagle's medium (DMEM) | Gibco | 11995065 | |
| Collagenase Ⅱ | Sigma-Aldrich | C6885 | |
| PBS | Gibco | 14190250 | |
| fetal bovine serum (FBS) | Gibco | 16000044 | |
| 50 mM ascorbic acid-2-phosphate | Sigma-Aldrich | A5960 | |
| Antibiotic-Antimycotic solution | Gibco | 15240062 | |
| 4% formaldehyde | Bio-solution | BP031 | |
| Triton X-100 | Sigma-Aldrich | X100-100ml | |
| BSA | Rdtech | C0082 | |
| TWEEN 20 | Sigma-Aldrich | P9416-100ml | |
| MKX (C-5) | Santa cruz biotechnology | sc-515878 | |
| Tenomodulin (N-14) | Santa cruz biotechnology | sc-49325 | |
| Fluorescence Mounting Medium | DAKO | S3023 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | |
| WST-1 | Dojindo Molecular Technologies | CK04 | |
| BrdU Cell Proliferation Assay Kit | Cell Signaling Technology | #6813 | |
| TRIzol Reagent | Invitrogen | 15596018 | |
| iScript cDNA Synthesis Kit | Bio-Rad | 170-8891 | |
| TaqMan Gene Expression Master Mix | Applied Biosystems | 4369016 | |
| GAPDH | Thermo Fisher Scientific | Hs02786624_g1 | |
| COL3A1 | Thermo Fisher Scientific | Hs00943809_m1 | |
| ACTA2 | Thermo Fisher Scientific | Hs00426835_g1 | |
| TAC1 | Thermo Fisher Scientific | Hs00243225_m1 | |
| TACR1 | Thermo Fisher Scientific | Hs00185530_m1 | |
| PTGS2 | Thermo Fisher Scientific | Hs00153133_m1 | |
| ACTB | Thermo Fisher Scientific | Hs99999903_m1 | |
| Cell Strainers (100 µm) | Corning | 352360 | |
| 100mm culture dish | Thermo Fisher Scientific | 8188207 | |
| 8-well Chamber Slide | Thermo Fisher Scientific | 154534 | |
| 96 Well Clear Flat Bottom Polystyrene TC-Treated Microplates | Corning | 3596 | |
| Nikon Eclipse 50i Microscope | Nikon | ||
| VERSA max microplate reader | Molecular Devices | ||
| CFX96 Real-Time PCR Detection System | Bio-Rad | ||
| Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128 | |
| Paraffins | Leica Biosystems | 3801340 | |
| Ethanol | JUNSEI CHEMICAL | 90303-2185 | |
| Hematoxylin | DAKO | CS70030-2 | |
| Eosin | DAKO | CS70130-2 | |
| Alcian blue | DAKO | AR16011-2 | |
| Citric acid | Sigma-Aldrich | 251275 | |
| Xylene | JUNSEI CHEMICAL | 25165-0430 | |
| Endogenous peroxidases | DAKO | S200380-2 | |
| Canada balsam | JUNSEI CHEMICAL | 23255-1210 | |
| Microtome Blade | FEATHER | A35 | |
| Slide glass | SUPERIOR | 1000612 | |
| Cover glass | Marienfeld-Superior | 101050 | |
| VEGF | Santa cruz biotechnology | sc-7269 | |
| SPSS Software | IBM | Ver. 18.0 | |
| Multi-purpose Centrifuge | LABOGENE | 1248R |
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Citar este artículo
Han, S., Kim, H. K., Ahn, J., Lee, D. H., Baek, M., Ye, G., Lee, J., Min, K., Oh, C., Lee, S. A Protocol to Acquire the Degenerative Tenocyte from Humans. J. Vis. Exp. (136), e57634, doi:10.3791/57634 (2018).